Ketone body 3-hydroxybutyrate enhances adipocyte function

Ketone bodies, including 3HBA, are endogenous products of fatty acid oxidation, and Hmgcs2 is the first rate-limiting enzyme of ketogenesis. From database analysis and in vivo and in vitro experiments, we found that adipose tissue and adipocytes express Hmgcs2, and that adipocytes produce and secrete 3HBA. Treatment with 3HBA enhanced the gene expression levels of the antioxidative stress factors, PPARγ, and lipogenic factors in adipose tissue in vivo and in adipocytes in vitro, accompanied by reduced ROS levels. Knockdown of endogenous Hmgcs2 in adipocytes markedly decreased 3HBA levels in adipocytes and decreased the gene expression levels of the antioxidative stress factors, PPARγ, and lipogenic factors with increased ROS levels. Conversely, overexpression of Hmgcs2 in adipocytes increased 3HBA secretion from adipocytes and enhanced the gene expression levels of the antioxidative stress factors, PPARγ, and lipogenic factors. These results demonstrate that 3HBA plays significant roles in enhancing the physiological function of adipocytes.

To validate the physiological regulation of 3HBA, wild-type C57BL/6 male mice were subjected to feeding or fasting conditions for 12 h, and then, blood samples and adipose tissues were collected. Body weight and blood glucose were decreased in the fasting group compared with the feeding group ( Fig. 1b and c), whereas the plasma 3HBA concentration was higher in the fasting group than in the feeding group (Fig. 1d), as previously reported 13  function of Hmgcs2 in vivo, we analyzed wild-type C57BL/6 male mice (Hmgcs2 +/+ ; WT) and Hmgcs2 homo knockout mice (Hmgcs2 −/− ; KO). Hmgcs2 of epiWAT were deleted in KO mice (Fig. 2a). WT mice and KO mice were fasted for 12 h followed by feeding for 12 h (Fig. 2b). The body weights of prefasting, postfasting, and postfeeding mice were unaltered between groups (Fig. 2c). Food intake, weight of adipose tissues, and blood glucose levels showed no significant differences between groups ( Fig. 2d-f). Recent studies have reported that antisense oligonucleotide of HMGCS2 caused insufficiency of ketogenesis resulting in accumulation of hepatic triacylglycerol 14 . In accordance with this report, liver weight of KO mice was heavier than WT mice (Fig. 2e). Plasma 3HBA concentrations at prefasting and postfasting were lower in KO mice than in WT mice (Fig. 2g). We previously reported that oxidative stress in adipose tissue is associated with adipose dysfunction 15 , and reduced oxidative stress by overexpression of antioxidative stress enzymes in adipose tissues resulted in adipose healthy expansion with enhanced lipogenesis 16 . We previously reported that 3HBA levels and mRNA expression of lipogenic factors were elevated in epiWAT of KKAy, a mouse model of obesity and type 2 diabetes, by dapagliflozin treatment 12 . Therefore, we measured oxidative stress-related gene expression in epiWAT. EpiWAT of the KO mice exhibited decreased transcript abundance of antioxidative stress factors, such as Foxo3 (Fig. 2h), SOD1 (Fig. 2i), and catalase (Fig. 2j), and adiponectin (Fig. 2k), a key adipocytokine in obesity-related disorders, which has antidiabetic, antiatherogenic, and anti-inflammatory properties 17 , compared with WT mice. Moreover, the expression levels of Scd1, a lipogenic gene, were decreased in epiWAT from KO mice compared to WT mice (Fig. 2l)  Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. www.nature.com/scientificreports/ 3HBA enhances the gene expression levels of antioxidative stress factors, PPARγ, and lipogenic factors in epiWAT in vivo. From these results (Fig. 2), we presumed that 3HBA might enhance mRNA expression levels of antioxidative-and de novo lipogenic enzymes in adipose tissues. To examine this hypothesis, wild-type C57BL/6 male mice were fasted for 12 h and then injected intraperitoneally with phosphate buffered saline (PBS) or 3HBA (20 mmol/kg of body weight) followed by feeding for 12 h (Fig. 3a). The body weights of prefasting, postfasting, and postfeeding mice were unaltered between PBS-and 3HBA-injected mice (Fig. 3b). Food intake, organ weight, and blood glucose showed no significant differences between groups ( Fig. 3c-e). Plasma 3HBA concentrations showed an increasing trend in 3HBA-injected mice compared to PBSinjected mice ( Fig. 3f; p = 0.2251). EpiWAT of the 3HBA-injected mice exhibited increased transcript abundance of antioxidative stress factors, such as SOD2 (Fig. 3g) and catalase (Fig. 3h), and PPARγ (Fig. 3i), a critical factor regulating lipid metabolism, adipocytokine secretion, and insulin sensitivity in mature adipocytes 18 , compared with PBS. Moreover, the expression levels of lipogenic genes, such as Acly (Fig. 3j), ACC (Fig. 3k), Fasn (Fig. 3l) and Scd1 (Fig. 3m) were increased in epiWAT from 3HBA-injected mice compared to PBS-injected mice. Collectively, these data demonstrate that single-bolus injection of 3HBA enhanced the mRNA expression levels of antioxidative stress factors, PPARγ, and lipogenic factors in adipose tissue.

3HBA exerts beneficial effects on adipocytes by reducing ROS via augmentation of antioxidative stress factors and inducing PPARγ, adiponectin, insulin signaling, and lipogenic factors in vitro.
In adipose tissues, the expression of lipogenic genes increases under refeeding conditions compared with fasting conditions 19 . In our mouse model, treatment with 3HBA induced an abundance of antioxidative stress factors, PPARγ, and lipogenic factors in adipose tissues under feeding conditions (Fig. 3). To further investigate the direct effect of 3HBA on adipocytes, we incubated 3T3-L1 adipocytes with or without 10 mM 3-HBA at a similar level in the plasma of fasting mice (Fig. 1d) in the presence of insulin to mimic feeding conditions. Treatment with 3HBA increased the gene expression of Hmgcs2 (Fig. 4a) and antioxidative stress factors, such as Foxo3, Mt2, SOD1, SOD2, and catalase, in 3T3-L1 adipocytes ( Fig. 4b-f). Foxo3 is a transcription factor that induces cell cycle arrest and resistance to oxidative stress 20   www.nature.com/scientificreports/ oxidative stress 16,21 . DCFDA is a cell-permeable fluorescent indicator for reactive oxygen species in living cells.
In parallel with the upregulation of antioxidative stress factors, the DCFDA assay indicated that treatment with 3HBA reduced reactive oxygen species (ROS) production in 3T3-L1 adipocytes (Fig. 4g). PPARγ is a critical transcription factor that maintains adipocyte function, such as insulin signaling and lipogenesis 22 , and we previously reported that ROS scavengers increased the expression of PPARγ and its downstream factor adiponectin and improved adipocyte function 15 . In accordance with these reports, treatment with 3HBA increased the mRNA expression of PPARγ and adiponectin ( Fig. 4h and i) and enhanced insulin-induced phosphorylation of Akt at serine 473 (pAkt) (Fig. 4j). Treatment with 3HBA significantly enhanced adiponectin secretion into the culture medium of 3T3-L1 adipocytes compared with those without 3HBA (Supplementary Figure S1). Furthermore, treatment with 3HBA increased the mRNA expression of lipogenic genes, such as Srebp1a, Acly, ACC, Fasn and Scd1, in 3T3-L1 adipocytes ( Fig. 4k-o). 3HBA significantly enhanced insulin-induced lipid accumulation measured by Oil red O stain in 3T3-L1 adipocytes (Fig. 4p). In this condition, 3HBA also induced glycerol secretion into the culture medium of 3T3-L1 adipocytes compared with those without 3HBA (Supplementary Figure S2), suggesting that lipogenic activity is induced more than lipolytic activity resulting in lipid accumulation by 3HBA treatment. However, treatment with 3HBA showed no change in the abundance of key lipogenic proteins, such as ACC, Fasn or Scd1, in 3T3-L1 adipocytes (Fig. 4q). Together, these data demonstrate that 3HBA, at physiological concentrations, exerts beneficial effects on adipocytes, accompanied by reduced ROS levels, via augmentation of antioxidative stress factors and induction of PPARγ, adiponectin, insulin signaling, and lipogenic factors in vitro.
Adipocytes express Hmgcs2 and produce and secrete 3HBA. To reveal the physiological regulation of ketogenesis-associated genes in adipose tissues (Fig. 1a), we compiled microarray datasets of upregulated genes by fasting in epididymal adipose tissue (epiWAT) of mice (GSE46495) 23 . In this dataset, the expression levels of Hmgcs2 and Hmgcl were significantly higher in epiWAT of fasting mice than in feeding mice. Sorting 4,666 upregulated genes in descending order, Hmgcs2 was among the most highly ranked genes (Fig. 6a). In epiWAT of fasting mice, mRNA expression of Hmgcs2 was elevated in comparison to feeding mice (Fig. 6b), consistent with analysis of microarray datasets (Fig. 6a). Next, to reveal the regulation of the Hmgcs2 gene in vitro, we incubated 3T3-L1 adipocytes with 3-HBA to mimic fasting conditions in vitro. Hmgcs2 transcript levels were strengthened by 3HBA treatment (Fig. 6c). To further reveal the hormonal regulation of Hmgcs2, 3T3-L1 adipocytes were treated with insulin or dexamethasone. Gene expression of Hmgcs2 was downregulated by insulin and markedly upregulated by dexamethasone ( Fig. 6d and e). Considering that plasma insulin levels were decreased whereas plasma 3-HBA and dexamethasone levels were increased under fasting conditions, the gene expression of Hmgcs2 is reasonably regulated by these factors in adipocytes.
Moreover, we compared the expression of Hmgcs2 between 3T3-L1 preadipocytes and adipocytes. Gene and protein expression of Hmgcs2 was augmented in differentiated 3T3-L1 adipocytes relative to undifferentiated www.nature.com/scientificreports/ 3T3-L1 adipocytes ( Fig. 6f and g). Strikingly, we observed a marked elevation of 3HBA concentrations in both cell lysate and cell culture supernatants in differentiated 3T3-L1 adipocytes compared to undifferentiated 3T3-L1 adipocytes ( Fig. 6h and i), suggesting that 3HBA was endogenously produced and secreted from differentiated adipocytes. Together, these data demonstrate that Hmgcs2 is expressed in adipose tissues and adipocytes and that adipocytes produce and secrete 3HBA.
Hmgcs2 regulates antioxidative stress factors and ROS, PPARγ, adiponectin, and lipogenic factors in an autocrine manner by endogenous 3HBA production in adipocytes. As shown in Fig. 6, we confirmed the gene and protein expression of Hmgcs2 and endogenous production of 3HBA in 3T3-L1 adipocytes. Furthermore, 3HBA treatment increased Hmgcs2 mRNA abundance in adipocytes (Fig. 4a). Based on these findings, we hypothesized that adipocyte-derived 3HBA acts in an autocrine manner to control antioxidative stress factors, PPARγ and lipogenesis factors in adipocytes. To confirm this hypothesis, we estimated the autocrine action of Hmgcs2 in 3T3-L1 adipocytes. We knocked down Hmgcs2 by siRNA followed www.nature.com/scientificreports/ by insulin treatment. The gene expression of Hmgcs2 was reduced by siRNA against Hmgcs2 (Fig. 7a). Under this condition, intracellular 3HBA levels were reduced (Fig. 7b). Additionally, knockdown of Hmgcs2 decreased the gene expression of antioxidative stress factors such as Foxo3, Mt2, SOD1, SOD2, and catalase ( Fig. 7c-g) and increased ROS production, as detected by DCFDA assay, in 3T3-L1 adipocytes (Fig. 7h). Furthermore, knockdown of Hmgcs2 reduced the gene expression levels of PPARγ, adiponectin ( Fig. 7i and j) and lipogenic factors such as Srebp1a, Acly, ACC, Fasn, and Scd1 (Fig. 7k-o) in 3T3-L1 adipocytes. Taken together, these data demonstrate that Hmgcs2 regulates antioxidative stress factors, ROS, PPARγ, adiponectin, and lipogenic factors in an autocrine manner by endogenous 3HBA production in adipocytes.

Discussion
In the current study, we found that adipose tissue and adipocytes express Hmgcs2 which is upregulated by 3HBA and dexamethasone, and downregulated by insulin, and that adipocytes produce and secrete 3HBA. Moreover, 3HBA plays significant roles in adipocytes to enhance the gene expression levels of antioxidative stress factors, PPARγ, and lipogenic factors with reduced ROS levels, probably in an endocrine and autocrine/paracrine manner (Fig. 8). In addition, Hmgcs2 gene expression was increased by fasting in adipose tissues of mice, suggesting the physiological role of endogenous ketogenesis of adipocytes in vivo. Previously, we reported that oxidative stress in adipose tissue suppresses the gene expression of PPARγ 15 . In addition, we established adipose ROS-eliminated and adipose ROS-augmented mice through the genetic manipulation of antioxidant-related genes. Adipose ROS-eliminated mice exhibited white adipose expansion with elevated lipogenic genes and improved insulin signaling. Conversely, adipose ROS-augmented mice exhibited restricted adipose expansion with reduced lipogenic genes and accelerated insulin resistance, indicating www.nature.com/scientificreports/ the significant role of ROS in regulating lipogenic genes and insulin signaling in adipose tissues 16 . On the other hand, it was reported that treatment with 3HBA epigenetically induced antioxidative enzymes in the kidney through HDAC inhibition, resulting in reduced ROS 9 . In the current study, we indicated that 3HBA reduced ROS, increased expression of PPARγ and lipogenic genes, and enhanced insulin signaling in adipocytes. One possible explanation is that the effect of 3HBA on enhancing adipocyte function might be attributed to reduced ROS levels through induction of antioxidative enzymes by epigenetic regulation. Ketone bodies, including 3HBA, are not only an alternative energy source under starvation but also play multiple roles as signaling molecules 1 . For instance, 3HBA induces histone b-hydroxybutyrylation, a recently described epigenetic mark that is involved in metabolic regulation in the liver 11 , and augments the expression of adiponectin in adipocytes 12 . In the intestine, a ketogenic diet increased serum and intestinal 3HBA levels to robustly oscillate in a circadian manner. This oscillation of 3HBA was coupled to tissue-specific cyclic histone deacetylase (HDAC) activity and histone acetylation, resulting in nuclear accumulation of PPARα, a metabolic master regulator, in the intestine 24 . On the other hand, 3HBA acts as a ligand of the G protein-coupled receptor (GPR). 3HBA is a ligand of GPR41 and regulates sympathetic nervous system activity and energy expenditure 25 . 3HBA is also a ligand of GPR109A and suppresses lipolysis through activation of immune cells 26,27 . However, gene expressions of both GPR41 and GPR109A in epiWAT of mice were not detected (GSE46495) 23 . Thus, ketone bodies, including 3HBA, exert various effects through multiple molecular mechanisms. Nevertheless, few studies have been reported on the relationship between 3HBA and adipocytes. Further studies are required to determine the mechanism by which 3HBA enhances the physiological function of adipocytes.
During fasting, circulating insulin levels are low, and the primary energy source shifts from glucose to free fatty acids (FFAs) released from adipocytes by lipolysis. FFAs are converted to ketone bodies mainly in the liver and transported to extrahepatic tissues. Therefore, prolonged fasting is accompanied by low serum insulin levels, elevated serum ketone bodies, and reduced storage of triglycerides in adipocytes. In the initial phase of feeding following prolonged fasting, plasma insulin levels rise quickly, while plasma 3HBA levels fall slowly and remain high in human subjects 28 . In the current study, 3HBA did not induce PPARγ or lipogenic genes in the absence of insulin (Supplementary Figure S3) but increased them accompanied by lipid accumulation in the presence of insulin (Figs. 3 and 4). In addition, 3HBA intensified insulin signaling (Fig. 4). In the current study, 3HBA induced lipid accumulation without changes in lipogenic protein expressions (Fig. 4). Insulin activates and ROS inhibits lipogenic activity 29,30 . In addition, insulin increases glucose uptake, and glucose is a substrate for lipogenesis. Moreover, 3HBA itself is a substrate for lipogenesis. Taken together, 3HBA potentiates lipid accumulation possibly by these multiple mechanisms to restore excessively mobilized and exhausted adipose tissue after prolonged fasting.
In conclusion, our results revealed for the first time that adipocytes express Hmgcs2 and produce 3HBA and that 3HBA enhances adipocyte function in endocrine and autocrine/paracrine manners. Further investigations are required to reveal the physiological and pathological significance of ketogenesis in adipocytes in vivo by establishing an adipocyte-specific Hmgcs2-deficient mouse model. Circulating 3HBA is primarily synthesized in the liver and secreted. In adipocytes, Hmgcs2 is upregulated by 3HBA and dexamethasone (Dex) but downregulated by insulin. 3HBA enhances the function of adipocytes by reducing fat ROS by inducing antioxidative stress factors and intensifying PPARγ, de novo lipogenesis, and insulin signaling in an endocrine (thick white arrow) and autocrine/paracrine manner through endogenous Hmgcs2 expression followed by the synthesis of 3HBA in adipocytes (thick black arrow).

Methods
Animal studies and approval. Seven-week-old male C57BL/6 J mice were obtained from CLEA Japan (Tokyo, Japan) and acclimated for more than 1 week before the experiment. Mice were housed individually in sterile cages, maintained in a room under controlled temperature (23 ± 1.5 °C) and humidity (45 ± 15%) on a 12-h dark/12-h light cycle, and had free access to water and normal chow diets (MF; Oriental Yeast, Tokyo). Samples from six tissues were collected, including epididymal fat, subcutaneous fat, mesenteric fat, BAT, liver, and skeletal muscle (gastrocnemius muscle). The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Osaka University, Graduate School of Medicine. These mouse studies were approved by the Ethics Review Committee for Animal Experimentation of Osaka University, Graduate School of Medicine, and carried out in accordance with the Institutional Animal Care and Use Committee Guidelines of Osaka University. WT (Hmgcs2 +/+ ) mice and KO (Hmgcs2 −/− ) mice were previously established by Hiroshi Maegawa's lab 31 . Briefly, a mouse model harboring a deletion mutation in exon 2 of the mouse Hmgcs2 gene was generated using the CRISPR/Cas9 system. The following sequences were used for single guide RNA (sgRNA) synthesis: sgRNA1, TGG AAC GCA CAA AGC TGC CG; sgRNA2, GTG CCT GCA GTG GTA CAG A. Cas9 mRNA and sgRNAs (Cas9 mRNA: gRNA1gg:gRNA2cc = 2:1:1) were microinjected into fertilized C57BL/6 J mouse embryos. Among these, one mouse line with a 38-bp deletion mutation in exon 2, which led to a frameshift and subsequent complete deletion of Hmgcs2 protein synthesis, was selected. Homozygous Hmgcs2 −/− mice were born from heterozygous intercrosses and used for phenotypic analyses in parallel with age-and sex-matched wild-type littermates as a control group. These mice were housed in a temperature-controlled environment (23 °C) with a 12-h light and 12-h dark (20:00-08:00) photoperiod and cared for in facilities operated by the Research Center for Animal Life Science at Shiga University of Medical Science. CLEA Rodent Diet (#CE-2, CLEA Japan) was used as the standard chow in the center. The experimental protocol was approved by the Gene Recombination Experiment Safety Committee and Research Center for Animal Life Science (RCALS) at Shiga University of Medical Science.
In the feeding versus fasting experiment, 10-week-old male C57BL/6 J mice were subjected to feeding or fasting conditions for 12 h and then anesthetized with isoflurane, and blood samples were collected from the heart, followed by sacrifice. Body weight was measured at the start and end of the experiment.
In the WT (Hmgcs2 + / +) mice versus KO (Hmgcs2 −/−) mice experiment, 12-or 13-week-old male mice were subjected to fasting condition for 12 h, followed by feeding for 12 h, and then anesthetized with isoflurane and blood samples were collected from the heart, followed by sacrifice. Body weight, blood glucose, and blood 3HBA were measured at prefasting, postfasting, and postfeeding, and food intake was determined by weighing the metal cage top, including the food at the end of the experiment.
In the injection of PBS or 3HBA experiment, 8-week-old male C57BL/6 J mice were subjected to fasting conditions for 12 h before being injected intraperitoneally with PBS or 3HBA (20 mmol/kg of body weight), fed for 12 h, and then anesthetized with isoflurane. Blood samples were collected from the heart, followed by sacrifice. Body weight was measured at prefasting, postfasting, and postfeeding, and food intake was determined by weighing the metal cage top, including the food at the end of the experiment.
Measurements of blood parameters. Blood glucose levels were measured by tail vein sampling before anesthesia using the Glutest Neo alpha (Sanwa Kagaku Kenkyusho, Nagoya, Japan). Blood 3HBA levels were measured by tail vein sampling before anesthesia using the PRECISION XCEED (Ketometer) (Abbott, Tokyo, Japan). Plasma concentrations of 3HBA were measured using a beta Hydroxybutyrate (beta HB) Assay Kit (Abcam) according to the instructions provided by the manufacturer.
Measurement of 3HBA abundance in cell lysate and culture supernatant. 3-HBA abundance in cell lysate and culture supernatant was measured using a beta Hydroxybutyrate (beta HB) Assay Kit (Abcam) according to the instructions provided by the manufacturer.
Effects of 3HBA on 3T3-L1 adipocytes. On day 7 after differentiation, the medium of 3T3-L1 adipocytes was replaced with DMEM composed of 2.5 mM glucose with 10 mM concentrations of 3-hydroxybutyric Doxycycline-inducible overexpression of Hmgcs2 on 3T3-L1 adipocytes. On day 5 after 3T3-L1-TetON-empty and 3T3-L1-TetON-Hmgcs2 adipocytes were differentiated, the adipocytes were treated with 2 µg/mL doxycycline for 48 h. On day 7 after differentiation, these adipocytes were maintained in serum-free DMEM composed of 25 mM glucose and 1 nM insulin for 24 h, followed by harvesting on day 8 after differentiation.
RNA extraction, cDNA synthesis, and quantitative real-time PCR. Total RNA was isolated with TRI-Reagent (Sigma-Aldrich) based on the method recommended by the manufacturer. cDNA was synthesized using Transcriptor Universal cDNA Master Mix (Roche) and was subjected to RT-PCR using FastStart Essential DNA Green Master Mix (Roche) on a LightCycler® 96 Instrument (Roche) according to the protocol provided by the manufacturer. The primers used are described in Supplementary Table 1.
Statistical analysis. All data were expressed as the mean ± SEM values. Differences between two groups were examined for statistical significance by Student's t-test. A P value < 0.05 denoted the presence of a statistically significant difference.

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.